Project Summary/Abstract The epithelial Na channel (ENaC) forms a pathway for Na+ absorption in the kidney, lung, and other epithelia. In order to maintain Na+ homeostasis and control blood pressure, ENaC is tightly regulated to respond to conditions of Na+/volume depletion and Na/volume excess. However, defects in this regulation are responsible for nearly all of the known inherited forms of hypertension, and contribute to the pathogenesis of cystic fibrosis. Thus, our long term objective is to understand the mechanisms that regulate ENaC as a prerequisite for the development of targeted treatments for these diseases. A recent convergence of discoveries has focused attention on mechanisms that regulate ENaC gating. In the biosynthetic pathway and at the cell surface, proteases cleave the extracellular domains of and ENaC, converting inactive channels into their active Na+-conducting form. Moreover, Na+ regulates ENaC gating through extracellular (Na+ self-inhibition) and intracellular (Na+ feedback inhibition) mechanisms to maintain homeostasis. Other extracellular molecules also regulate ENaC activity. However, there are critical gaps in our knowledge about the molecular mechanisms and channel structures that underlie this regulation. A critical advance is the very recent solution of the crystal structure of a closely related channel, ASIC1. This has provided an unprecedented look at the structures that may underlie the regulation of gating of the DEG/ENaC ion channel family. Taking advantage of these advances in the understanding of ENaC gating and the ASIC1 crystal structure, the overall goal of this proposal is to understand structure-function relationships that regulate ENaC gating. We propose three Specific Aims. 1. In preliminary studies, we discovered that intracellular Na+ regulates ENaC by altering proteolytic cleavage of and ENaC. In this aim, we will test the hypothesis that Na+ alters cleavage by inducing a conformational change in the ENaC extracellular domain. We will also identify the ENaC sequences are required. 2. ENaC is exposed to extremes of pH in the kidney and lung. In preliminary studies, we found that ENaC activity is regulated by extracellular pH. In this aim, we will investigate the molecular mechanisms and identify the ENaC sequences that are required for pH to regulate ENaC. 3. ENaC is also exposed to significant changes in Cl- concentration. Our preliminary work indicates that Cl- modulates ENaC current and is required for Na+ self-inhibition, a mechanism by which extracellular Na+ regulates ENaC. In this aim, our goal is to understand the mechanism(s) by which Cl- alters ENaC current, and to identify residues in the extracellular domains that mediate this effect. By using innovative approaches and by testing novel hypotheses, this work will provide a new understanding of mechanisms that regulate ENaC gating, and hence, epithelial Na transport and Na homeostasis.